Production of substances rich in riboflavin



Patented Feb. 27, 1945 2,370,177 PRODUCTION OF SUBSTANCES RICH IN RIBOFLA VIN David A. Legg and Samuel C. Beesch, Philadeln a, Alcohol Pa., assignors to Publicker Commercial Company, Philadelphia, Pa., a corporation of Pennsylvania No Drawing. Application September 3, 1941, Serial No. 409,378

11 Claims.

The present invention relates to the production of high-vitamin-content concentrates from bacterial fermentations and it relates more particularly to the production of concentrate having a high riboflavin-content from butanobacetone fermentations.

An object of the present invention is to provide a fermentation-residue concentrate from butanolacetone fermentation flavin-content. I

Another object of the present invention is to provide a method for producing high riboflavincontent concentrate from butanol-acetone fermentation.

Still another object of the present invention is to provide a method of fermenting .mash by means of butanol and acetone producing bacteria which will give a fermentation residue having a high riboflavin-content.

-Other objects and purposes of the present invention are apparent in the following specification and appended claims.

It has long been known that the residue obtained from the fermentation of a starchor sugar-containing mash by butanol-and-acetoneproducing bacteria contains beneficial substances which make it purposes.

Thus, United States Patent No. 1,587,101 to Woolner described the salvaging of this fermentation slop or residue and the concentration and drying thereof for use as stock feed.

Nelson, Heller & Fulmer writing in the Journal of Industrial & Engineering Chemistry (February 1925, page 199 et seq.) determined the vitamin B content of molasses.

Booher writing in the Journal of the American which has a high ribomercial production of high riboflavin-content concentrates from such fermentations, was the fact that the riboflavin-content of the final concentrate varied considerably from batch to batch.

We have found that the riboflavin-content of the fermentation residue and of the final dried concentrate can be increased and made more uniform by modifying the fermentation as will be hereinafter described;

The present invention is preferably directed to the fermentation of starchy or cereal mashes by bacteria which will be referred to as Clostridium acetobutylicum. The name Clostridzum acetobutylz'cum as employed in the specification and claims is not intended to designate any single organism, but is used to identify the group of organisms which are proteolytic in character and are capable of breaking down the proteins to form compounds having amino groups and which are capable of fermenting fermentable solutions to produce butanol and acetone as defined in United States Patent No. 1,911,411 to Viljoen.

We have found that the addition to cereal or starchy mashes (either before orafter sterilizaparticularly suitable for feeding Medical Association (volume 110, No. 4, page 1105) describes early work on riboflavin as .obtained from various enzymatic processes.

Pett writing in the Biochemical Journal .(volume 29, 1935, page 937) describes the bacterial production of riboflavin (employing the synonymous term lactoflavin).

For the past five years or more, it has been well known in the art of butanol-acetone fermentations that the fermentation residue and the final concentrate obtained by evaporating,

drying and grinding said residue, has a higher riboflavin content than can be accounted for tion thereof) of any compound which, in aqueous solution liberates a sulphite ion, greatly improves the riboflavin-content of the dried fermentation residue and makes the riboflavin-content of successful fermentation residues more uniform.

We have found that the amounts of the sulphite-liberating material to be added are limited by the toxicity of the sulphite ion or of the positive ion to the organism under conditions of fermentation.

As used in the specification, the expressions "cereal or star-ch31" mashes indicate aqueous suspensions of such cereals as rice or corn which have been cooked to gelatinize the starch but which have not been saccharifled to any appreciable extent, although it has been found that a small amount of sugar may be present without interfering with results.

Riboflavin-contents are expressed in terms of gamma; a gamma being defined as one mi cro-gram of riboflavin per gram of substance.

- The dry matter making up the cereal or starchy mashes employed usually assays less than 10 gamma of riboflavin before fermentation.

We have found that the addition, for example, to some of them at the start of fermentation and of sodium sulphite is particularly effective in in- 1 to others 24 hours later. The flasks were inocucreasing and making more uniform the riboflavinlated with a culture of Clostridium acetobutulicontent of the dried fermentation residue. cum and incubated for '10 hours at 37C. The In the case of sodium sulphite, which we prefer clear layer of fermented mash was evaporated to use, the effective percentage in a mash having to dryness in vacuo and the residue assayed for 4 to of cereal lies between the more or less riboflavin with the results shown in Table II.

approximate limits of 0.005% to 0.05% of the anhydrous salt. The preferred amount of sodium 1 I Table II sulphite is between the more or less'approximate 1o limitation of 0.014 to 0.035 gram per 100 cc. of Riboflavin fermentation mash. I micrograms We have found that amounts of sodium sul-m, r .Medium f g 23 3;

phite below this range may not be fully effective, p tatiou residue while amounts above this range may be too in-' 5 gz ytofermentation. Y a 7 I N e amounts of other compo nd's containing 2 e m t s p ion y be b s d on't e pe cent o '2 12123332.""*"'*"I 3133i? 3% 33333112 Hi3 SO: and we have successfully used such com- 23 g ag? 24 pounds as potassium sulphite, sodiumhydrogen 0.007% added 24 11rd. 1,000 sulphite, barium sulphite, sodium thiosulphate; H Mmstm' ethyl sulphite, ammonium sulphite, and potassium pyro sulphite. From the results shown in Table n it would seem prefer t dd s p t0 t r lo that the addition of sodium sulphite at the beeterehy mesh before Sterilization. thereoflginning of fermentation ismore effeotive'than thoushitmay beadded after sterilization 0r u the addition after theiermentation has proin! the first few hours of fermentationgrossed for 24v hours and that such addition The following ereexamples-of how our P s t should be made either before the .start of the f maybe preeticemw i1 fermentation or at some time earlier than 24' j EXAMPLE 1 l 3!: hours after the fermentation has set in. These experiments also indicate that 0.014% ofsodium mmmm Rmmmm sulphite is more eiiective than 0.001%.

PRODUCTION FROM RICE MASH Fsmviarira'riori t A mash was made up containing-5 grams of polished rice and 0.043 gram of sodium sulphite per 100 cc. 'Ihemash containing the sulphite was sterilized for 1 /2 hours at pounds of steam pressure in an autoclave. "A similar control mash Flasks of 5% cereal mash were made up with was made up without the sulphite'. 'The flasks various amounts of sulphites and allied comof mash were fermented under identical condii0 pounds, h s mashegcontained rice and tions by a strain of Clostridium acetobutylicum. 25%- com nd were sterilized for 1 /2 hours at for 66 hours at 37 C. 'Thedecantedliquor from 5 pounds f steam pressure, Aft 7 hours the ft:rmeiitation m W85 Evaporated to fermentation at 37 C. by a strain of Clostridium Insurance or SUiLrriIrr: Ion in Forms 0mm ram Somrm SULPHITE ness in vacuo at temperatures not exceeding 100 wn m the settled clear liquor was 0- The driedfresidue from the mash wh vaporated in vacuo to dryness and the residue had been treatedwith sulphiteeeeeyed 8 assayed for riboflavin. The average result of of riboflavin while the. residueinom the three flasks under each condition is shown in trol mash (which contained no sulphite) assayed Table-11L t t Y Y 3'75 gamma 01 riboflavin. In anothersimilar ex- "I perlment, the l'esultsshown in Table I were 0b-. .7

Table! i Riboflavin l m 0 Riboflavin "\Mmm Bowmwmid firl i ler- H i n. .(miqoartml I p l ,mantntion V Perce'ntofsodium sulphite' 5. u r fi' ffi g I v residue tation v residue 1 A 2. 5% rice, 2.5%'0orn.. 1.180 1? 13 ".'.do. 1,800

I 0 1,500 None 580 D V 1,z;0 0.014. 1,650 E 4-.500 00:0. 1 1,400 I ,1

' would Thus, thetresults set are in Tahl e'iwould sei n indicate thatsodium sulphite is the. most eilecw indicate a interminncentrationf e to tive'and that'sodi uml thiosiilphate s theleast on w Sodium p tel e emewliei f Wtl't 9 fective-ofthe sources oif l hite, 1031.

and0.03% of thefermentationmash; r. Amm -11 Flasks oi' steriieimash'-c0ntainin8;2 5%.01:90 iii th r eipn wqy s,ma e

ished rice and 2.5% of com were made up and varisulphite and other compounds containing a sulous amounts of sterile sodium sulphite were added 1 phite ion with the results shown in Table IV.

nuance orisutrnirr irn Ferris Oriiriir'rrum i EXAMPLE -V mrwmcn or Sunrn'rrr Ion Wars: rm: Frauenrsrron Mesa Un'rsssrgo Grvss A Msxnmu Ymo Rice-com mash of suitable composition will without addition of sulphites, give a fermentation residue assaying from 350 to 2000 gamma. The influence of sulphite appears to assure an average yield closer to 2000 rather than to raise the yield appreciably above the otherwise-obtained maximum of 2000 gamma. In this ex periment mashss of 2.5% rice and 2.5% corn with and without sulphite were fermented for 66 hours at 37 C. with a strain of Clostridium acctobutulicum with the results shown in 'l'a--.

mash with Clostridium acctobutvlicum;

ble V. f

Table V Riboflavin micrograms 0 Medium Snlphits as per cent in mash g' mentation residue 244% rice, 2.5% corn--- Nil (control) 2, 050 Do 0.02% potauium sulpbite---. 1, 980 Do I 0.02% sodium sulphite 2. 180

While certain specific embodiments of the present invention have been described hereinahove, such embodiments are illustrative and not restrictive and the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof.- It is therefore desired that references be had to the appended claims rather than to the foregoing description to indicate the scope of the present invention.

Having thus-described our invention, we hereby claim and desire to secure byLetters Patent" the following:

capable of liberating sulphite radical in the presence of water.

3. A process for producing substances having generally uniform relatively high riboflavin content including the step of fermenting an aqueous cereal mash with C'lostridium acetobutylicum in the presence of 0.005 to 0.05% of a substance capable of liberating sulphite radical in the presence of water.

4. A process for producing substances having generally uniform relatively high riboflavin content including the step of fermenting an aqueous cereal mash with Clostridium acetobutulicum in the presence of 0.014 to 0.035% of a substance capable of liberating sulphite radical in the presence of water.

5. A process for producing substances having generally uniform relatively high riboflavin content which comprises fermenting an aqueous cereal mash with Clostridium acetobutylicum and adding to the mash within 24 hours of the start of fermentation a small amount of a substance capable of, liberating sulphite radical in the presence of water.

GPA process for producing substances having generally uniform relatlvelyhigh riboflavin content including the steps of fermenting an aqueous cereal mash with Clostridium acetobut'ulicum in the presence of a small amount of a substance capable of liberating sulphite radical in the presence of water and recovering concentrates rich in riboflavin from the fermentation residue.

-'7. A process for producing substances having generally uniform relatively high riboflavin content including the steps of fermenting an aqueous cereal mash with Clostridium qcetobutylicum in the presence of a small amount of a substance capable of liberating sulphite radical in the presence of water, distilling-off volatile fermentation products, and concentrating and drying the fermentation slop, thereby to produce a concentrate rich in riboflavin.

8. A process for producing substances generally uniform relatively high riboflavin content which comprises preparing an aqueous mash having generally equal parts of corn and rice, adding 0.005% to 0.05% of a substance capable of liberating sulphite radical in the presence of water, and fermenting the mash with Clostridium acetobutulicum.

9. A process for producing substances having generally uniform relatively high riboflavin content including the step of fermenting an aqueous cereal mash with Clostridium acetobutylicum in Y the presence of a small amount of pyrosulphite.

1. A process for producing substances'having of a substance capable of liberating sulphite radical in the presence of water and fermenting, the

Y 2. A process for producing substances having generally uniform relatively high riboflavin content including the step of fermenting an queous cereal mash with Clostfidium acetobutulicum in the presence ofa small amount of a substance 10. A process for producing substances having generally uniform relatively high riboflavin content including the step of fermenting an aqueous cereal mash with Clostridium acetobutulicum in the presence of'a small amount of ethyl sulphite.

11. A process for producing substances havin generally uniform relatively high riboflavin con tent including the step of fermentingan aqueous cereal mash with Clostridium acetobutulicum in the presence of a small amount of sodium sulphitc.-

LEGG.

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